Note: Descriptions are shown in the official language in which they were submitted.
133967~
~OLYAMIDE RESIN AND RESIN-PROTIDE, AND METHODS FOR PREPARATION
OF REAGENTS AND OF I~MUNODIAGNOSTIC TESTING
BACKGROUND OF THE INVENTION
- The present invention relates to the synthesis and use of
synthetic peptides and proteins to induce an immune response in
experimental animals. More particularly, the present invention
relates to a polyamide resin, a method of making that polyamide
resin, a method of inducing an immune response in an experimental
animal using a conjugate of a peptide or protein synthesized on
that resin, and the the use of the resin for immunodiagnostic
purposes.
Solid phase peptide~synthesis is a valuable tool for inves-
tigating the structure and mechanism of action of proteins. Most
such synthetic methods involve the use of a cross-linked poly-
styrene based resin as the solid phase to which the peptide is
anchored during assembly, usually through a linker molecule.
Assembly is accomplished by a repetitive cycle of adding a
~rotected amino acid to the solid phase, selectively removing
(deprotecting) a protective group on that amino acid, and adding
additional suitably protected amino acids (for a review, see
Merrifield, R.B., ~Solid-phase Peptide Synthesisn, 32 Adv.
Enzymology 221 (1969)).
Although cross-linked, polystyrene based resins are most
commonly used as supports in solid phase peptide synthesis, their
relatively hydrophobic character in comparison to the polar
organic media required to solubilize reactants can be problematic
in peptide chain assembly. Such media may freely solvate the
growing peptide, yet incompletely swell the polystyrene matrix.
~3~fi7a
Within the polymer lattice, impaired diffusion of reagents and
steric hindrance can contribute to lowered efficiency during
S coupling cycles, which, on a repeated basis, lowers final yields
appreciably. During the early stages of assembly, when the resin
7 to peptide mass ratio is high and the physical properties of the
8 support dominate, this lowered efficiency is particularly acute,
- 9~ - Those shortcomings led to the development of a cross-linked,
~10 polydimethylacrylamide based support which is highly polar in
- ~ character and is freely permeated by the requisite solvents for
- ~12 peptide synthesis. Atherton, E., D.L.J. Clive and R.C. Sheppard,
13 ~Polyamide Supports For Polypeptide Synthesisn, 97 J. Amer. Chem.
-I Soc. 6584 (1975); Arshady, R., E. Atherton, M.J. Gait, K. Lee and
lS R.C. Sheppard, "Easily Prepared Polar Support For Solid Phase
l6 Peptide And Oligonucleotide Synthesisn. 1979 J.C.S. Chem. Comm.
17 425 11979). The polyamide resin, as the amino methyl derivative,
18 can accommodate synthetic schemes incorporating alternate pro-
19 tection strategies through selection of the appropriate linker
molecule, which links the C-terminal residue to the support. The
2I peptide or protein thus synthesized, which will be referred to
22 throughout the present disclosure as a "protide~, can be used in
23 a number of investigative applications.
24 Of particular interest to the present invention is the use
2S of the protide as an immunogen. It has previously been
26 demonstrated that synthetic peptides analogous to sequences
27 contained in viral encoded proteins have proven useful for
28 identification of native antigen determinants associated with
29 such proteins. Several laboratories have reported studies on the
antigenic activity of various HBsAg synthetic peptides.
3I Dreesman, G.R., et al., 295 Nature 158 (1982); Lerner, R.A., et
32 al. 78 Proc. Natl. Acad. Sci. USA 3403 (1981); Prince, A.M., et
33 al., ~9 Proc. Natl. Acad. Sci. USA 579 (1982). The induction of
34
01/MRW12 -3-
133~70
an antibody response to HBsAg, using such peptides,
proved to be relatively weak, but could be enhanced
through coupling of peptides to a carrier protein prior
to immunization. Lerner, et al., supra; Sanchez, y., et
al., 18 Intervirology 209 (1982). Because the prediction
of potential antigenic determinants of immunogenic
proteins based on primary sequences analysis is not
exact, the identification of putative epitopes through
trial and error can be laborious. A method which
involves the delineation of native antigenic sequences
with synthetic peptides which does not require
purification of the synthetic peptide and coupling of the
peptide to carrier proteins offers significant
advantages. It is, therefore, an object of an aspect of
the present invention to provide a method of preparing a
polyamide resin upon which a protide can be synthesized
using solid phase synthetic methods which can be injected
into an experimental animal to induce an immunogenic
response without separation of the protide from the
resin.
It is another object of an aspect of the present
invention to provide a polyamide resin for solid phase
protide synthesis, and a conjugate of that polyamide
resin and synthesized protide, which can be injected into
an experimental animal to induce an immunogenic response.
It is another object of an aspect of the present
invention to provide a polyamide resin-protide conjugate
for use in in vitro immunological assays.
Another object of an aspect of the present invention
is to provide a method of preparing a polyamide resin for
solid phase protide synthesis comprising cross-linking a
dimethylacrylamide monomer with a molecule containing a
functional group by free radical co-polymerization in an
aqueous solution which has been emulsified by a detergent
when added to an organic solvent medium.
133~670
Another object of an aspect of the present invention
is to provide a method of inducing an immunogenic
response in an experimental animal with synthetic peptide
or protein comprising preparing a polyamide resin,
synthesizing a peptide or protein on that polyamide
resin, and immunizing an experimental animal with the
polyamide resin-synthetic peptide or synthetic protein
conjugate.
Another object of an aspect of the present invention
is to provide an assay for detection of proteins such as
antigens and antibodies using the polyamide resin-protide
conjugate of the present invention.
Another object of an aspect of the present invention
is to provide a method of inducing an immunogenic
response in an experimental animal using the polyamide
resin-protide conjugate of the present invention.
Another object of an aspect of the present invention
is to provide a polyamide resin for solid phase protide
synthesis which does not require the separation of the
protide from the resin and he subsequent purification of
the protide before the use of that protide to, for
instance, induce an immunogenic response.
Another object of an aspect of the present invention
is o provide a polyamide resin for solid phase protide
synthesis which is particularly useful in the mapping of
the antigenic determinants of a protein as a result of
the elimination of the steps of separation of the protide
from the resin and the subsequent purification of the
protide before the use of the protide in a binding assay.
These and other aspects and advantages of the
present invention will be clear to those skilled in the
art from the following description.
D
1 3 3 ~ b 7 U
SU~n~ARY OF THE INVENTION
This invention provides a method of preparing a
polyamide resin for solid phase protide synthesis, comprising
adding a dimethylacrylamide monomer to an alkenylamine
monomer and a cross-linker in aqueous solution;
emulsifying the aqueous solution in an organic
solvent;
adding an initiator and a promotor to co-polymerize
and cross-link the monomers and the cross-linker. The beads are
then isolated and used as the solid phase for synthesis of a
peptide.
Also provided is a polydimethylacrylamide resin
including the following characteristics. Occupies about
2.5 times its dry bed volume when swollen in methylene
chloride; about 8% to about 12% cross-linked; contains
about 50 to about 400 micromoles allylamine per gram of
resin; capable of being substituted with about 0.1 to
about 0.5 micromoles of amino acid per gram of resin when
coupled through an amide bond to the resin; and lacking
20 unreacted free amine when loaded. -
Also provided is a pharmaceutical composition for
use in inducing an immunogenic response in a mammal which
comprises an effective amount of a polyamide resin-
protide conjugate, together with a pharmaceutically
acceptable carrier therefor.
Also provided is a method of inducing an immunogenic
response in an experimental animal comprising preparing a
polyamide resin, synthesizing a protide on the polyamide
resin, and immunizing an experimental animal with the
polyamide resin-protide conjugate.
An in vitro diagnostic assay is also provided which
comprises preparing a polydimethylacrylamide resin,
synthesizing a protide on the polyamide resin to form a
polyamide resin-protide conjugate; contacting the
polyamide resin-protide conjugate with a body fluid
~'
133~7f~
suspected of containing antibodies capable of binding
specificaliy to the protide; and detecting the bound
antibodies. The bound antibodies are then detected using,
for instance, an enzyme linked immunosorbent assay.
An ln vitro diagnostic assay product is also
provided which comprises a polydimethylacrylamide resin
including the following characteristics:
occupies about 2.5 times its dry bed volume when
swollen in methylene chloride;
is about 8~ to about 12~ cross-linked;
contains about 50 to about 400 micromoles
allylamine/g of resin;
is capable of being substituted with about 0.1 to
about 0.5 micromoles amino acid/g resin when coupled
through an amide bond to the resin;
lacks unreacted free amine when loaded; and
a protide conjugated to said polyamide resin.
A method of preparing polydimethylacrylamide resin
beads is also provided, comprising
mixing allylamine monomer with dimethylacrylamide
monomer and cross-linker in an aqueous solution;
emulsifying the mixture in an organic solvent to promote
the formation of polymer resin beads of a desired size;
adding an initiator and a promotor to polymerize the
allylamine monomer, dimethylacrylamide monomer and cross-
linker into polydimethylacrylamide resin beads; and
isolating the thus formed beads.
~
/
- 6a -
~;
~,
~ 133~73
DETAILED DESCRIPTION OF THE INVENTION
As noted above, the term "protiden, as used herein, refers
- to both the peptides and proteins which are synthesized according
~ to the method of the present invention. A significant advantage
-~ ~ of the method of the present invention is that the protide syn-
''' --~~ ~ ~~ thesized on the polyamide resin can be used to induce an immuno-
. . .
genic response in a mammal without being separated from the resin
~- and purified.
Il The usual method of coupling the protide to polystyrene
~ 12 based resins is through a benzyl ester derivative, and separation
- of the protide from the resin is usually accomplished by either
- acidic or basic cleavage. Benzyl esters are susceptible to
several such methods of cleavage, but are also stable throughout
- ~ the multiple deprotection, neutralization and coupling reactions
l7 which are characteristic of solid phase synthetic methods.
18 Hydrazine has also been used to separate the protide from the
, _ ___
--- resin (Kessler, W. and B. Iselin, 49 ~elv. Chim. Acta 1330
(1966)) as have various ammonolytic (MAnning, M., 90
21 J.Am.Chem.Soc. 1348 (1968)) and other methods. However, those
- 22 methods all require that appropriate steps be taken to avoid
r, 23
damage to the protide followed by purification of the protide
24 from the byproducts of the synthesis, including amino acids,
short peptides, decomposition products of the resin, and
26 sometimes, peptides containing incompletely removed protecting
27 groups. Although purification can sometimes be accomplished by a
28 direct crystallization, in syntheses in which the contaminating
29 peptides are of approximately the same size and composition as
the desired product, more selective techniques must be employed.
31 Regardless of the method of separation and purification, those
32 requirements add time-consuming steps to the synthesis which
33 often lower the total yield of protide. The method of the
34
01/MRW12 -7-
~:
present invention requires no such separation and purification,
thereby decreasing the amount of time required to accomplish the
synthesis and raising the protide yield.
The polyamide resin of the present invention is prepared by
cross-linking a commercially available dimethylacrylamide monomer
in aqueous solution using a diaminoalkane, preferably a
diaminoalkane having alkenoyl groups at either end of the
molecule such as N,N'-bis-alkenoyl-diaminoalkane. In a presently
preferred embodiment, the cross-linker is either N,N'-bisacrylyl-
1,3-diaminopropane or N,N'-bisacrylyl-1,3-diaminobutane prepared
according to the method of Halpern and Sparrow (J.A. Halpern and
J.T. Sparrow, "An Improved Procedure For The Synthesis Of N,N'-
bisacrylyldiaminoalkanes", 10 Synthetic Comm. 569 (1980)). The
use of the propane analog is preferred because it yields a
polymer of larger pore size and improved swelling properties
during protide synthesis than the polymer obtained by use of the
ethyl analog. However, it will be understood by those skilled in
the art who have the benefit of this disclosure that the other
diaminoalkanes listed in that report, N,N'-bisacrylyl-1,2-
~20 diaminoethane and N,N'-bisacrylyl-1,6-diaminohexane, as well as
~~ ~ other diaminoalkanes, are also appropriate for use in the
preparation of the resin of the present invention.
, ,
A functional monomer is included in the cross-linked resin
of the present invention. The term "functional monomer" refers
to those alkenyl amines which are used to anchor the C-terminal
amino acid of a synthetic protide to the resin. The functional
monomer, when protected with the methylsulfonylethyloxycarbonyl
(MSC) group (see Tesser, G.I. and I.C. Balvert-Geers, "The
Methylsulfonylethyloxycarbonyl Group, A New And Versatile Amino
Protective Function", 7 Int. J. Peptide Protein Res. 295 (1975)),
-
--8
. ,.~
~:i ' ' k .
, . ~ 1. ,. ~. .,',', ' - ,
:
~39~70
is referred to as an MSC alkenyl amine. Those functional
monomers are prepared by reaction of the commercially available
chloride derivative with the alkenylamine, and the MSC protective
group is subsequently removed with base. However, the MSC group
is not required. The polyamide resin of the present invention is
8 also prepared by simply adding an excess of the allylamine,
9~ followed by filtering or other method to remove the resulting
r 10 fines. The amount of functional monomer added is selected to'
1l yield a resin substitution of between about 0.1 mmol and about
l2 0.5 mmol per gram of resin, and preferably in the range of about
13 0.2 mmol to about 0.4 mmol per gram of resin. The initiator can
14 be any of the initiators known to those skilled in the art such
as a persulfate or riboflavin, and is preferably ammonium
16 persulfate.
17 Because the above-described substances are combined in aque-
18 ous solution, they are collectively referred to as "the aqueous
19 phase~. The next step in the preparation of the polyamide resin
of the present invention is to combine the aqueous phase with an
21 organic phase. The term ~organic phase" refers to an organic
22 solvent which, when combined with the aqueous phase and stirred,
23 results in a suspension from which the resin of the present
24 invention is obtained. In a presently preferred embodiment, the
2S organic phase comprises a mixture of hexane and carbon tetra-
26 chloride.
27 An emulsifier is added during the stirring to allow for the
28 formation of beads of uniform size. The emulsifier can be any
29 detergent known to those skilled in the art, and in a presently
preferred embodiment, is either sorbitan sesquioleate, sorbitan
3I monolaurate or sorbitan monodecanoate. The amount of detergent
32 added is adjusted to give a spherical resin of approximately
33 uniform size. A decrease in the amount of detergent resylts in
34 ,
01/MRW12 -9-
1~ ~9~70
~-.
; 2-
- an emulsion which yields increased amounts of~Iarger, amorphous
material, which could contribute to a reduction to the inte~nal
- growing chains of amino acids. An increase in the amount of
~ 7 detergent increases the amount of fine material, which is
-~ difficult to remove without the loss of significant amounts of
the resin. Those fines clog the reaction vessels of the peptide
synthesizer as well as the associated lines and valves.
A promoter is then added to promote the polymerization of
the monomers in the suspension, resulting in the formation of
beads of the polyamide resin of the present invention. A number
13 of promotors are known to those skilled in the art, but
14 particular success in preparing the polyamide resin of the
IS present invention has been obtained with
N,N,N',N'-tetramethylethylenediamine (TEMED). The resulting
1' beads are then filtered and washed, the MSC group (if present) is
18 removed with base, and the beads are dried. The beads may then
19 be sifted through a mesh sieve to insure relatively uniform size.
Overall yields using the method of the present invention ranged
21 from about 87% to about 94% from starting monomers.
22 The aminomethyl, cross-linked polydimethylacrylamide resin
23 of the pr-~sent invention provides maximum exposure of the protide
24 in an aqueous solution, and the resin-polymer backbone does not
2S restrict the protide conformationally. The exposure of the
26 protide is the result of the ability of the polyamide resin to
27 swell to many times its dry bed volume when highly solvated by
28 water.
29 Protides are then synthesized on the beads by coupling to a
linker which is attached to the resin with an activator. The
31 term "linker" refers to a linking group which links the carboxyl
32 group of the first amino acid of the protide to the polymeric
~- 33 resin. In the presently preferred embodiment, this linker is an
34
0l/MRWl2 -l0-
~ , :' r- J
: i 3~70
1 oxyalkyl benzoic acid (OBA) to which an amino acid residue is
2 coupled to serve as the first amino acid in the protide chain.
3 Because the OBA linker is used to attach the C-terminal amino
4 acid to the polyamide resin of the present invention, anhydrous
hydrogen fluoride can be used to remove the side chain protecting
6 groups from the protide without significant loss of the protide
7 from the resin. In the below-described examples, the amino acid
8 of choice is glycine, which is protected with the t-butyloxy-
- 9 carbonyl (t-Boc) protecting group, but it will be understood by
those skilled in the art who have the benefit of this disclosure
11 that the amino acid could be any amino acid, particularly, the
12 amino acid which is the first amino acid in the protide to be
~- 13 synthesized, and that other protecting groups are equally suit-
14 able. The glycine residue serves the additional function of a
spacer between the protide and the resin-polymer backbone.
16 The Boc-glycyl-4-(oxymethyl) benzoic acid which is the
17 presently preferred linker was prepared by a modification of the
18 method described by Mitchell, et al. (Mitchell, A.R., S.B.H.
19 Kent, M. Engelhard and R.B. Merrifield, A new synthetic route to
tert-butyloxycarbonylaminoacyl-4-(oxymethyl) phenylacetamido-
21 methyl-resin, an improved support of solid-phase peptide syn-
22 thesis, 43 J. Org. Chem. 2845 (1978). An important
~i,,
23 modification of the Mitchell, et al., method is the elimination
; 24 of the use of dimethylformamide as a solvent. That solvent is
difficult to evaporate, consequently, even though evaporation can
26 be hastened by raising the temperature, the method is still
27 time-consuming. The activator used to couple the linker to the
28 polyamide resin prepared as described above is diisopropyl
29 carbodiimide and 4-dimethylaminopyridine, but it will be
understood by those skilled in the art that other activators such
. ~ .
7 ~
r
- as dicyclohexylcarbodiimide and 4-methylpyrrolindinopyridine are
~- equally suitable for such a purpose.
After synthesis of the protide on the polyamide resin, the
'- 6 polyamide resin-protide conjugate is used for a number of pur-
poses, including in vitro assays, inducing an immunogenic re-
sponse in experimental animals, or mapping antigenic
determinants. For instance, an in vitro assay is conducted by
crushing the beaded polyamide resin-protide conjugate with a
1I mortar and a pestle and absorbing the crushed conjugate onto a
12 solid phase such as a microtiter test plate with neutral pH
13 buffer. Serum or other body fluid suspected of containing an
14 antibody capable of specifically binding the protein or peptide
on the resin is then incubated with the absorbed conjugate,
16 unbound antibodies are removed by washing, and the bound
17 antibodies are detected by enzyme linked immunosorbent assay,
18 biotin-avidin amplified assay or other detection methods such as
- 19 are known in the art.
The polyamide resin-protide conjugate can also be used to
21 map antigenic determinants by simply removing a portion of the
22 polyamide resin-protide conjugate at intervals during the synthe-
23 sis of the protiae, deprotecting the protide, and testing each
24 removed portion in serial fashion to determine that point in the
2S 6ynthesi 3 at which the protide binds antibody. This method is
26 made possible by the elimination of the separation and purifica-
27 tion steps required in other synthetic methods. The conjugate
28 can also be tested for its ability to bind antibody by crushing
29 and absorbing to a solid support such as a microtiter test plate
and assayed as described above. Separation of the protide from
the resin and purification of the protide is not required for
32 such an assay.
33
34
01/MRW12 -12-
b 7 a
The polyamide resin-protide conjugate is also useful as an
immunogen. The conjugate is used directly for immunization of
experimental animals with or without an adjuvant. The term
"experimental animal", as used herein, refers to any animal
capable of an immune response. The experimental animals of
primary interest are mammals, but an immunogenic response can be
induced in other experimental animals such as birds using the
method of the present invention. For instance, an immune
response specific for hepatitis B, as measured by radioimmuno-
assay, was induced by immunization of rabbits using a conjugate
- :,,~
comprised of a synthetic peptide with the same sequence as the
hepatitis B antigen (HBsAG) peptide 119-159 emulsified in
Freund's complete adjuvant. Similar results, as measured by
- radioimmunoprecipitation, were obtained with a conjugate
comprising a peptide corresponding to the protein coat of the
AIDS virus HTLV-III and the polyamide resin of the present
invention.
A particularly valuable embodiment of the invention is a
polyamide resin including the following characteristics:
occupies about 2.5 times its dry bed volume when swollen in
methylene chloride;
about 8% to about 12% cross-linked;
,--
~ ~- contains about 50 to about 400 micromoles allylamine per
gram of resin;
capable of being substituted with about 0.1 to about 0.5
micromoles of amino acid per gram of resin when coupled through
,,
an amide bond to the resin; and
lacking unreacted free amine when loaded.
The polyamide resin so obtained is preferably in the form of
beads such that the beads are less than about 180 microns in
diameter.
- As a further preferred feature of the invention we provide
-13-
~ '''"'f
3 ~ ~ 7 i~
an in vitro diagnostic assay product comprising a polyamide
resin including the following characteristics:
occupies about 2.5 times its dry bed volume when swollen in
methylene chloride;
about 8~ to about 12~ cross-linked;
contains about 50 to about 400 micromoles allylamine per
gram of resin;
capable of being substituted with about 0.1 to about 0.5
micromoles of amino acid per gram of resin when coupled through
an amide bond to the resin;
lacking unreacted free amine when loaded; and
a protide conjugated to said polyamide resin.
ccording to a further feature of the invention we provide a
pharmaceutical composition for use in inducing an immunogenic
response in a mammal which comprises an effective amount of a
polyamide resin-protide conjugate, together with a pharmaceuti-
cally acceptable carrier therefor.
As another embodiment of the invention we provide an in
vitro diagnostic assay method comprising preparing a polyamide
resin, synthesizing a protide on the polyamide resin to form a
polyamide resin-protide conjugate, contacting the polyamide
resin-protide conjugate with a body fluid suspected of contain-
ing antibodies capable of binding specifically to the protide and
detecting the bound antibodies.
~ The present invention can be better understood by reference
_ to the following examples, which are presented for purposes of
exemplification and not limitation.
EXAMPLE I
PREPARATION OF FUNCTIONAL MONOMER
Five grams of (26.8 mmol) 2-methylsulfonylethyloxycarbonyl
chloride (MSC chloride) (K+K Labs, ICN) were dissolved in 15 ml
acetonitrile and added dropwise over a 20 minute period to a
stirred solution of 2.1 ml (28 mmol) redistilled allylamine
; -13a-
1~3~70
(Kodak) and 4.9 ml (28 mmol) redistilled diisopropylethylamine
(DIEA) in 20 ml acetonitrile. (DIEA (Aldrich) was refluxed over
~- ninhydrin and redistilled.) The solution was stirred an addi-tional two hours and the solvent evaporated. The residue was
;,; taken up in 250 ml ethyl acetate and allowed to stand for one-two
~ -13b-
: ~3~9~7a
~, "
hours. The bullc of the DIEA hydrochloride salt precipitated as
needles. After filtration and evaporation, the crude material
S was dissolved in a minimal amount of chloroform and loaded onto a
6 silica gel G-60 column (60 g) packed in the same solvent. Elu-
7 tion with chloroform yielded pure MSC-allylamine. (RF on TLC =
8 .64 (Solvent = CHCl3: CH30H, 9:l).)
9~~ The remaining DIEA salts adsorbed to the column under these
10 conditions. Occasionally, material migrating near the solvent
front on TLC contaminated the MSC-allylamine column fractions.
12 That material was removed by crystallizing the MSC allylamine
13from methylene chloride-hexane at -20~C. Yield was 4.8 g (8~%
4 from MSC chloride).
EXAMPLE II
16PREPARATION OF CROSS-LINKER
1~The cross-linker N,N'-bisacrylyl-l,3-diaminopropane was
18 prepared according to the method set out in Helpern and Sparrow,
19 supra. Briefly, diaminopropane (Aldrich) was dissolved in ace-
20 tonitrile and added dropwise to an acrylyl chloride-acetonitrile
21 solution at 4~C, allowed to warm to room temperature and stirred.
22 The diaiminopropane dihydrochloride was removed by filtration,
23 washed with warm acetonitrile, and the combined filtrates were
24 concentrated in vacuo. N,N'-bisacrylyl-1,3-diaminopropane was
25 crystallized at 4~C overnight and the resulting plates filtered
26 and dried in vacuo.
27EXAMPLE III
PREPARATION OF POLYAMIDE RESIN
28In a glass, 2-liter cylindrical, fluted polymerization ves-
29 sel fitted with a nitrogen inlet and mechanically driven glass
30stirrer were added 490 ml hexane and 290 ml carbon tetrachloride.
31 The solution was purged for l5 minutes with nitrogen to remove
32 oxygen. To this solution was added an aqueous solution
33containing N,N'-bisacrylyl-1,3-diaminopropane (2.9 grar~s, 15.9
34
Ol/MRWl2 -14-
~ .
~ ~'3~7i~
. ~ ..
mmol) prepared as described in Example II mixed with 18.2 ml (175
- 4 mmoll of N,N-dimethylacrylamide (PolySciences). Ten g (48 mmoll
S MSC allylamine prepared as described in Example 1 and 120 ml
6 water were added, and the solution was filtered and degassed
7 before addition to the organic phase. The density of the re-
8 sulting mixture was adjusted to obtain a uniform suspension with
- 9c stirring at 400-450 RPM. Ammonium persulfate (BioRad) (0.5 g in
1 5 ml H2O) and 1 ml of either sorbitan sesquioleate or sorbitan
- ll monolaurate (Sigma) were added. A solution of 3 ml N,N,N',N'-
l2 tetramethylethylenediamine (TEMED) (BioRad) in 2 ml R2O, pH
l3 6.5-7.5 (conc. HCl) was then added to the suspension. The
l4 suspended emulsion was stirred for two hours under nitrogen
atmosphere. ~he resultant beaded material was then filtered and
l6 washed sequentially with water (one liter) methanol (one liter),
l7 a mixture of dioxane:methanol:2 N NaOH (14:5:1, two liters, to
l8 remove MSC group), water (two liters), 1 N HC1 (two liters),
l9 water (two liters), and then methanol (two liters). ~he resin
was defined by suspension in methanol and decanting (3x). After
21 swelling in methylene chloride (Baker HPLC grade), the resin was
22 shrunk in hexane and dried in vacuo. ~arge amorphous mater~al
- 23 was removed by sifting the resin through an 80 mesh (180 micron)
~ 24 sieve.
,,:,
The degree of functionalization was checked by coupling Boc-
26 alanine to 100 mg of the resin using diisopropylcarbodiimide as
27 activator and 4-dimethylaminopyridine (recrystallized from ethyl
28 acetate) as catalyst. Amino acid analysis showed a substitution
29 of 0.15 to 0.35 mmol/g resin, depending on the lot, and resins
were prepared with as little as about 0.1 and as much as about
3l 0.5 mmol/g resin depending upon the amount'of allylamine added.
32 ~he loaded resin gave no detectable staining with picryl-sulfonic
33 acid, indicating the absence of unreacted free amine. When
'.
01/MRW12 -15- '-
-
r
' ' ~
~33~7~
",
swollen in methylene chloride, the beads occupied about 2.5 times
their dry bed volume. When swollen in dimethylformamide or an
,". ~ S
; -,~J:~ ~r, f~,. .,,,~,,.,,~,.,~ aqueous solution, the beads occupied approximately four and six
-- times their dry bed volume, respectively.
EXAMPLE IV
g PREPARATION OF LINRER
The linker Boc-glycyl-4-(oxymethyl) benzoic acid was pre-
ll pared by modification of the method of Mitchell, et al., supra.
l2 Briefly, the 4-(bromomethyl) benzoic acid phenylacylester was
l3 prepared by dissolving 10.3 ml redistilled diisopropylethylamine
l4 and 12.05 g (60.6 mmol) bromoacetophenone in 450 ml ethyl ace-
tate. 4-(bromomethyl) benzoic acid (13.89 g, 60.6 mmol) was
16 added in seven equal portions over a three hour period to the
17 stirred solution at 40-50~C. Stirring was continued for two more
:
18 hours at room temperature. Precipitated Et3N HBr was removed by
l9 filtration and the ethyl acetate solution was washed four times
with 50 ml each of an aqueous solution of 10% citric acid,
2I saturated sodium chloride, saturated sodium bicarbonate, and
22 saturated sodium chloride. The organic phase was dried over
23 anhydrous magnesium sulfate and freed of solvent by rotary
24 evaporation under reduced pressure. The residue was crystallized
~ from CH2C12-petroleum ether (1:3 v/v) to give the 4-(brc ~.-thyl)
26 benzoic acid phenylacylester.
27 The 4-(bromomethyl1 benzoic acid phenylacylester was
28 converted to Boc-glycyl-4-(oxymethyl) benzoic acid by dissolv~ng
29 Boc-L-glycine (25 mmol, 4.38g) in 15 ml methanol and titrating to
neutrality with tetramethylammonium hydroxide ~25~ in methanol).
31 Solvent was removed azeotropically with chloroform in vacuo, and
32 the salt dissolved in 150 ml acetonitrile. To the stirred
solution was added 5.8 g (17.5 mmol) of the 4-(bromomethyl)
34
01/MRW12 -16-
~ IE .,. "~
-:
7 0
L
benzoic acid phenacyl ester prepared as described. After
~- ~ overnight mixing, the precipitated tetramethylammonium bromide
was filtered and the solvent evaporated. ~he residue was
~ 6
- - dissolved in 400 ml ethyl acetate and the solution filtered. ~he
-xi - ~ 7 organic phase was then washed successively with 10~ aqueous
- citric acid (3 x 75 ml), 0.5 M sodium bicarbonate: 0.5 M
potassium carbonate (2:1), pH 9.5 (8 x 75 ml), then water (3 x 75
ml). The aolution was dried (MgSO4) and the solvent removed in
vacuo. The residue was dissolved in 200 ml of 85% acetic acid to
which 23 g acid washed zinc dust was added. The mixture was
stirred until the phenacyl ester was no longer visible by TLC (4
14 - 5 hours). The zinc was filtered and washed with 50 ml acetic
acid, and the combined solutions were lyophilized. The residue
~- was suspended in 100 ml water:300 ml ethyl acetate, and the pH
~- 17
adjusted to 1.5 (conc. HCl). The aqueous layer was extracted
-~- '8
' with a second portion of ethyl acetate (200 ml), and the combined
19 extracts were washed with water (100 ml). After drying (MgSO4)
and evaporating, the Boc-glycyl-4(oxymethyl) benzoic acid was
21 purified by recrystallization from methylene chloride:hexane at
22 -10~. Yield was 4.5 g (14.5 mmol, 83% from the phenacyl ester).
23 EXAMPLE V
24 COUPLING OF LINKER TO POLYAMIDE RESIN
2S Boc-glycyl-4-(oxymethyl) benzoic acid prepared as described
26 in Example IV was coupled to the aminomethyl polyamide resin (1.2
27 g) prepa.ed as described in Example III on a Biosearch Sam II
= 28 automated peptide synthesizer using diisopropylcarbodiimide and
~~ 29 dimethylaminopyridine as activator in a 1:1 methylene
chloride:dimethylformamide solution. Both methylene chloride
31 (Baker HPLC grade) and dimethylformamide (Baker Photrex grade)
- 32 were used without further purification. Following treatment with
33 hydrogen fluoride, 50 mg of the glycyl resin was found to contain
34
1~ '
nl/~or.~ ~
9 g 7 0
-~ 0.13 mmol/g by amino acid analysis. Amino acid analysis was
performed using a Beckman Model 119 amino acid analyzer following
S
-- either a two hour hydrolysis (12 N HCl:propionic acid, 1:1,
135~C) or 24 hour hydrolysis 16 N HCl, 110~C) of resin bound
78 peptides.
EXAMPLE VI
g ~YNl~SIS OF HEPA~ITIS B ANTIGEN PE~TIDE
0 The hepatitis B surface antigen (HBsAg) peptide 119-159 was
1l assembled on the aminomethyl, cross-linked polydimethylacrylamide
l2 resin prepared as described in Example III, having the Boc-
13 glycyl-4-(oxymethyl) benzoic acid linker prepared as described in
14 Example IV attached thereto using the method described in Example
IS V, with all residues being double coupled using a Biosearch Sam
- l6 II automated peptide synthesizer. The sequence of that RBsAg
peptide is as follows, and is relative to the-AYW subtype:
l8 H2N-GLY-PRO-SER-ARG-THR-CYS-MET-THR-THR-ALA-GLN-
19 120
GLY-THR-SER-MET-~YR-PRO-SER-CYS-SER-C:YS-
21 1~ ~ S ~
22 THR-LYS-PRO-SER-ASP-GLY-ASN-CYS-THR-SER-
23
24 ILE-PRO-lLE-PRO-SER-SER-TRP-AlA-PHE-GLY~Resin~
2S
26 The peptide included the following substitutions to control the
2~ specific formation of disulfide loops: serine for cystéines 121,
28 138, and 149. The cysteines 139 and 147 sulfhydryls were blocked
29 by the 4-methoxybenzyl group, while the sulfhydryls of cysteines
at 124 and 137 were protected as the S-acetamidomethyl
~ 31 derivatives. ~-N-tBoc protected amino acids were purchased from
_- ~ 32 Bachem. Additional side chain protecting groups were as follows:
33 formyl group for the indole nitrogen of tryptophan; be~z~le~hers
.
34
01/MRWl2 -18-
-
39~70
for threonine and serine hydroxyls; acetamidomethyl or
-~ 4
4-methoxybenzyl for cysteine sulfhydryls as described above;
S
-~' benzyl esters for ~-carboxyl of aspartic acid and the ~-carboxyl
of glutamic acid: 2-chlorobenzyloxycarbonyl for ~-amino group
:, ' 7
of lysine; 2,6-dichlorobenzyl ether for the phenolic hydroxyl of
tyrosine; and the p-tosyl group for the quanidine of arginine.
For the synthesis, methylene chIoride (Baker HPLC grade) and DMF
(Baker Photrex grade) were used without further purification.
-.~; Diisopropylethylamine (DIEA) (Aldrich) was refluxed over nin-
~ ~ ~ hydrin and redistilled. Trifluoroacetic acid (Halocarbon) was
- 13 redistilled, with the middle cut used in deblocking steps. All
'other chemicals were reagent grade or better and used without
IS further purification.
Side chain protecting groups were removed from the completed
peptidyl-resin by treatment with anhydrous HF (20 ml/g resin) at
18 0~ for thirty minutes, containing 10~ anisole and 2% ethanedi-
thiol. Following evaporation of HF, the peptidyl-resin was
2 washed successively with ether, 1% acetic acid, methanol, 5~ DIEA
21 in methylene chloride, methanol, then 1% acetic acid. The pep-
22 tidyl-resin was dried in vacuo. The formyl group was ,~- -ved
23 from the tryptophan by treatment with ethanolamine at 0~. A
24 disulfide bridge was formed between cysteines 139 and 147 by
2S potassium ferricyanide treatment. A second disulfide bridge
26 between cysteines 124 and 137 resulted during simultaneous
2~ removal of the acetimidomethyl moieties with a solution of iodine
28 in acetic acid.
EXAMPLE VII
IN VITRO ASSAY FOR PRESENCE OF HBsAg ANTIBODY
31 Human serum can be assayed for the presence of antibody
32 specific for the HBsAg peptide 119-159 by the following in vitro
33 assay. A quantity of the HBsAg peptide 119-159-polyamide resin
34
01/MRW12 -19-
~ ~3~70
prepared as described in Example VI is crushed with a mortar and
pestle. A microscope may be used to verify that the polyamide
resin-peptide conjugate has been crushed. Approximately 100 ,ul
~- of a solution containing between about 200 nanograms and about 10
micrograms of the crushed polyamide resin-peptide conjugate in a
neutral pH buffer such as phosphate buffered saline (PBS) is
absorbed to a solid phase such as Dynatech Immunolon II
Microfilter test plate. Nonspecific binding sites are blocked
with 10% normal goat serum (NGtS) and the plate is washed with
Tween 20 PBS (T-PBS) to remove unbound antibodies.
Human sera suspected of containing antibodies specific for
HBsAg peptide 119-159 and rabbit antisera produced by i -~zing
rabbits with the polyamide resin-HBsAg peptide 119-159 conjugate
diluted in 10% NGtS i8 then added to the polyamide
resin-peptide-coated plate and incubated for one hour at 37~C,
followed by washing with T-PBS. Biotin goat anti-human IgG or
biotin goat anti-rabbit IgG (Vector ~aboratories, Burlingame, CA)
is then incubated with the bound human and rabbit sera,
respectively, for one hour at 37~C. The wells are washed and
avidin conjugated to horseradish peroxidase (Av-HRP) is added for
20 minutes at room temperature. The wells are then washed with
i T-PBS to remove an unbound Av-HRP and peroxidase activity i8
determined using a 1 mM solution of 1,2'-azino-di(3-ethyl-benz-
i thiazoline sulfonic acid) (Sigma Chemical Co.) and 0.03% H202 as
substrate. The reaction is stopped with 5% (w/v) sodium dodecyl
sulfate in water prior to quantitating spectrophotometrically at
410 nm using a Dynatech plate reader. Optimal dilutions of each
reagent are selected by titration. All reagents for dete~ ining
specific binding except the substrate are diluted in 10% NGtS.
," ',' ,
-20-
'- 1339~70
- EXAMPLE VIII
IN VITRO ASSAY FOR PRESENCE OF HBsAg ANTIBODY
Human serum was assayed for the presence of antibody to
6 hepatitis B surface antigen by the following in vitro assay. A
7 10% solution of the polyamide resin-HBsAg peptide 119-159
~8 conjugate was prepared in a buffered bovine serum albumin (BSA)
-~ g solution containing a final concentration of 40% tetrahydrafuran.
An equal volume of antibody specific for the HBsAg peptide~
ll 119-159 containing between 100,000 and 1,000,000 counts per
i12 minute I125 was added and incubated with gentle rocking. The
13 resulting suspension was centrifuged and the pellet washed with
- 14 1% BSA-Tween 20 PBS, then centrifuged again. The radioactivity
- IS of the pellet was then counted in a Gamma counter. The results
i.' '1," . .......
16 clearly indicate the recognition of the polyamide resin HbsAg
17peptide 119-159 conjugate by native HBsAg antibody:
18 Glycyl resinresin-HbsAg peptide
(Control)119-159 conjugate
l9 IgG Human anti-HB#l 1240 cpm* 22,840
2 IgG Human anti-HB~2 1921 28,732
~ Normal human IgG 1432 1949
21* all measurements in counts per minute
22EXAMPLE IX
23USE OF PO~YAMIDE RESIN-PROTIDE CONJUGATE TO
INDUCE AN IMMUNOGENIC RESPONSE IN MAMMALS
24The polyamide resin-peptide conjugate prepared as described
2S in Example VI was used to induce an immunogenic response in rab-
26 bits as follows. New Zealand white female rabbits were i n~zed
27 with three monthly intramuscular injections of either 200 ~g
28 HBsAg peptide 119-159 (as the peptide-resin conjugate) or only
29 glycyl-resin emulsified in Freund's complete adjuvant (range of
immunogen, 50 ug to 1 mg for rabbits). Serum was collected after
31 bi-weekly bleeding and checked for anti-HBsAg activity using a
32 commercially available radioimmunoassay (RIA) kit (AUSAB, Abbott
33 _
34
01/MRW12 -21-
~1 3 t3 '3 ~ 7 ~
Laboratories). The recognition of the native HBsAg surface
antigen by the anti-peptide 119-159 antibody response induced in
S the rabbits is demonstrated by the following data developed by
6 that RIA.
ANTIBODYa
8 TITER
(RIA UNITS
PER
RABBIT IMMUNOGEN IMMUNIZATIONMILLILITER)
10 No. 1 Glycine-Resin Preimmune a 58b
Il Primary ~8
Secondary ~8
l2 Tertiary <8
l3 No. 2 HBsAg Peptide-Resin Preimmune ~8
Primary ~8
l4 Secondary 183
Tertiary 920
l5 No. 3 HBsAg Peptide-Resin Preimmune ~8
l6 Primary <8
Secondary 72
l7 Tertiary 262
l8 ~ a. Sera obtained prior to immunization.
b. Antibody titer to HBsAg is below the sensitivity of the RIA
l9 kit and is considered not to contain specific antibodies.
As can be seen by this data, the polyamide resin-HBsAg peptide
2l 119-159 conjugate containing a single disulfide bridge between
22 cysteines 139 and 147, when used to immunize rabbits, yielded
23 anti-peptide antisera which cross reacted with HBsAg.
24 EXAMPLE X
2S SY~ SIS OF HTLV-III ANTIGEN PEPTIDE
26 The HTLV-III peptide gp 120 503-532 was assembled on the
27 cross-linked polydimethylacrylamide resin prepared as described
28 ln Example III, having the Boc-glycyl-4-(oxymethyl) benzoic acid
29 linker prepared as described in Example IV attached thereto using
the method as described in Example V, in the same method as de-
3I scribed for the synthesis of the HBsAG peptide 119-159 in Example
32 VI, the only difference being the order in which the protected
33
34
01/MRW12 -22-
~gG7 0
amino acids were added. The sequence of the HTLV-III peptide gp
4120 503-532 is as follows:
S
69p 120 503~532
H2N--YAL--ALA--PRO--THR--LYS--ALA--LYS--ARG--ARG--
7 ~o~
8VAL--VAL--GLN--ARG--GLU--LYS--ARG--ALA--VAL--GLY--
9 ~ - ILE--GLY--ALA--LEU--PHE--LEU--GLY--PHE--LEU--
GLY--ALA--GLY--O--CH2--~--Y--N--CH2--(RESIN)
5:~2 h
12 EXAMPLE XI
USE OF POLYAMIDE RESIN-HTLV-III S~Ni~r~IC PEPTIDE
CONJUGATE TO INDUCE AN IMMUNOGENIC RESPONSE
1 Rabbits immunized with the polyamide resin-HTLV-III peptide
lS 503-532 conjugate produced a specific anti-peptide response as
16 determined by an enzyme lin~ed immunosorbent assay conducted
l7 according to the method of Example VII (with the use of antisera
18 produced~ by immunizing the rabbits with the polyamide
l9 resin-HTLV-III peptide 503-532 conjugate rather than the
conjugate which included the HBsAg peptide 119-159 as used in
21 that Example). The results of that immunoassay are presented
22 graphically in Fig. 1. The data represented by the circles is
23 data from rabbits immunized with that conjugate, the data
24 represented by triangles is from those same rabbits before
2S immunization (the solid circles and triangles are both from one
26 rabbit; the open circles and triangles are both from one rabbit;
27 the open circles and triangles are both from a second rabbit). A
28 conjugate c~mprised of the polyamide resin and a third
29 peptide failed to demonstrate significant binding. One of
30 the rabbits produced an anti-HTLV-III response specific for the
3I gp 120 envelope protein of HTLV-III based on a
32 radioimmunoprecipitation assay conducted according to the method
33 of Allan, J.S., et al. 228 Science 1091 (1985) and Barin, ~., et
01/MRW12 -23-
1339~70
al., 228 Science 1094 (1985).
The ability of the rabbit antisera generated to
peptide 503-532 to neutralize HTLV-III infectivity was
assessed on the basis of a reduction of reverse
transcriptase activity using a tenfold dilution of the
HTLV-III stock with a constant amount of antisera.
Barre-Sinoussi, F., et al., 220 Science 868 (1983). A
single rabbit anti-peptide 503-532 antiserum efficiently
reduced HTLV-III replication at day 10 compared to pooled
human sera from AIDS patients at tenfold dilutions of
virus. A second rabbit antiserum to that peptide failed
to reduce HTLV-III replication and so was used as a
control throughout the RT assay. No anti-HTLV-III
activity was detected in this particular antiserum based
on radioimmunoprecipitation even though the rabbit
received a similar immunogen and produced a detectable
anti-peptide response.
The antiserum that neutralizes HTLV-III detected
both gp 120 and gp 160 envelope glycoproteins. This
rabbit antiserum was found to be less efficient in
neutralizing HTLV-III compared to human AIDS serum on day
12 and 15 following HTLV-III infection.
The foregoing examples are presented for purposes of
exemplification of the method of the present invention.
Variations in those methods will be known to those
skilled in the art, and it is expected that all such
variations will be made without departing from the spirit
and scope of the present invention as claimed in the
following claims.
- 24 -
S
